Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation

Multinucleated giant cells (MNGC) derived from avian blood monocytes presen t, like osteoclasts, an unusual cytoskeletal organization characterized by (1) cortical rings of actin filaments, (2) unique adhesion structures calle d podosomes and (3) vinculin containing focal complexes which are not visib ly connected to F-actin structures, The Rho family of small GTPases plays a n essential role in the regulation and organization of cellular cytoskeleta l structures including F-actin and vinculin associated structures. Using ba cterial toxins such as modified exoenzyme C3 (C3B) and toxin B or overexpre ssion of constitutively active Rac and Rho proteins fused to the green fluo rescent protein (GFP), we show that Rac and Rho play antagonistic roles in regulating the morphology of osteoclast-like cells. Inhibition of Rho by C3 B triggered MNGC spreading whereas activated Rho promoted cell retraction. However, inhibition or activation of Rho led to complete disorganization of fibrillar actin structures, including podosomes. Toxin B inhibition of Rho , Rac and Cdc42 induced a time dependent F-actin and vinculin reorganizatio n. Initially. actin fibers with associated adhesion plaques formed and disa ppeared subsequently. Finally, only small focal complexes remained at the M NGC periphery before retracting. At the time when actin fibers formed, we o bserved that Rac was already inhibited by toxin B, By combining C3B treatme nt and overexpression of a dominant negative form of Rac (N17Rac), we show that the formation of these focal adhesion and actin fiber structures requi red neither Rho nor Rac activity. Moreover, our results show that podosomes are extremely unstable structures since any modifications of Rho or Rac ac tivity resulted in their dissociation.